Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

Abstract

BackgroundMammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements.MethodsTumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content.ResultsIndividual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0.20 Cqs accompanied by >94 % concordant single marker assignments for all four markers. In platform 2, the inter-site SD estimates were between 0.40 and 0.66 Cqs while the concordance for single marker assignments was >94 % for all four markers. The agreement reached between the two qPCR systems located in one site was 100 % for ERBB2, 96.9 % for ESR1, 97.2 % for PGR and 98.6 % for MKI67. RT-qPCR for individual markers was stable up to a 64-fold dilution for a typical clinical sample. There was no change in assay performance detected at the level of individual markers or subtypes after using different RNA isolation methods. The presence of up to 80 % of surrounding non-tumor tissue including in situ carcinoma did not affect the assay output. Sixteen out of 20 RNXtract eluates yielded more than 50 ng/μl of RNA (average RNA output: 233 ng/μl), whereas DNA contamination per sample was restricted to less than 15 ng/μl. Median recovery rate of RNA extraction was 91.0 %.ConclusionsIn this study the performance characteristics of MammaTyp...

An international reproducibility study validating quantitative determination of ERBB2, ESR1, PGR, and MKI67 mRNA in breast cancer using MammaTyper®

“…These data therefore confirm the high analytical performance of the MammaTyper® that was previously reported in the original technical validation of the test [21] but was shown in this study in a more comprehensive and challenging methodological setting. In our study, ten different laboratories were able to generate consistent and highly concordant test results after an initial training and a relatively short familiarization period.…”

“…Primary analysis outputs are the normalized, quantitative single-marker results given as 40 −∆∆Cq (quantification cycle) values on a continuous scale [21]. The test also provides the status of each marker as a binary category (positive or negative) based on clinically validated marker- and device-specific cutoff values.…”

Evaluation of the prognostic value of CD3, CD8, and FOXP3 mRNA expression in early‐stage breast cancer patients treated with anthracycline‐based adjuvant chemotherapy

“…The quality and quantity of RNA were checked by measuring CALM2 expression as a surrogate for amplifiable mRNA by qRT‐PCR. CALM2 was used as endogenous reference, as it had previously been identified as being highly and stably expressed among breast cancer tissue samples . Of the 975 FFPE tumor tissue samples collected, 857 (87.9%) had enough material left for RNA isolation needed for this study.…”

“…No‐template controls were assessed in parallel to exclude contamination. The qRT‐PCR method has recently been validated …”

FOXM1 overexpression is associated with adverse outcome and predicts response to intravesical instillation therapy in stage pT1 non‐muscle‐invasive bladder cancer

“…The mRNA expression levels of FOXM1, KRT5, KRT20 and MKI67, as well as one reference gene, namely, calmodulin (CALM2), were determined by quantitative RT-PCR, which involves reverse transcription of RNA and subsequent amplification of cDNA executed successively as a one-step reaction using Taqman Primer/Probes. The robustness and usefulness of CALM2 as a housekeeping gene for diverse candidate genes has been demonstrated in multiple publications by our group [18,19] and resulted its introduction as a housekeeping gene in communaut e europ eenne-certified in vitro diagnostics products such as Endopredict [20].…”